Project description:The plasmid pRN1 encodes for a multifunctional replication protein with primase, DNA polymerase and helicase activity. The minimal region required for primase activity encompasses amino-acid residues 40-370. While the N-terminal part of that minimal region (residues 47-247) folds into the prim/pol domain and bears the active site, the structure and function of the C-terminal part (residues 248-370) is unknown. Here we show that the C-terminal part of the minimal region folds into a compact domain with six helices and is stabilized by a disulfide bond. Three helices superimpose well with the C-terminal domain of the primase of the bacterial broad host range plasmid RSF1010. Structure-based site-directed mutagenesis shows that the C-terminal helix of the helix bundle domain is required for primase activity although it is distant to the active site in the crystallized conformation. Furthermore, we identified mutants of the C-terminal domain, which are defective in template binding, dinucleotide formation and conformation change prior to DNA extension.
Project description:The molecular motor protein myosin VI moves toward the minus-end of actin filaments with a step size of 30-36 nm. Such large step size either drastically limits the degree of complex formation between dimer subunits to leave enough length for the lever arms, or requires an extension of the lever arms' crystallographically observed structure. Recent experimental work proposed that myosin VI dimerization triggers the unfolding of the protein's proximal tail domain which could drive the needed lever-arm extension. Here, we demonstrate through steered molecular dynamics simulation the feasibility of sufficient extension arising from turning a three-helix bundle into a long ?-helix. A key role is played by the known calmodulin binding that facilitates the extension by altering the strain path in myosin VI. Sequence analysis of the proximal tail domain suggests that further calmodulin binding sites open up when the domain's three-helix bundle is unfolded and that subsequent calmodulin binding stabilizes the extended lever arms.
Project description:The SAP domain from the Saccharomyces cerevisiae Tho1 protein is comprised of just two helices and a hydrophobic core and is one of the smallest proteins whose folding has been characterised. ?-value analysis revealed that Tho1 SAP folds through a transition state where helix 1 is the most extensively formed element of secondary structure and flickering native-like core contacts from Leu35 are also present. The contacts that contribute most to native state stability of Tho1 SAP are not formed in the transition state.
Project description:We identify a distinctive circular dichroism (CD) signature for self-assembled 14-helical beta-peptides. Our data show that self-assembly leads to a mimimum at 205 nm, which is distinct from the well-known minimum at 214 nm for a monomeric 14-helix. The onset of assembly is indicated by [theta]205/[theta]214>0.7. Our results will facilitate rapid screening for self-assembling beta-peptides and raise the possibility that far-UV CD will be useful for detecting higher-order structure for other well-folded oligoamide backbones.
Project description:The mechanisms governing atlastin-mediated membrane fusion are unknown. Here we demonstrate that a three-helix bundle (3HB) within the middle domain is required for oligomerization. Mutation of core hydrophobic residues within these helices inactivates atlastin function by preventing membrane tethering and the subsequent fusion. GTP binding induces a conformational change that reorients the GTPase domain relative to the 3HB to permit self-association, but the ability to hydrolyze GTP is required for full fusion, indicating that nucleotide binding and hydrolysis play distinct roles. Oligomerization of atlastin stimulates its ability to hydrolyze GTP, and the energy released drives lipid bilayer merger. Mutations that prevent atlastin self-association also abolish oligomerization-dependent stimulation of GTPase activity. Furthermore, increasing the distance of atlastin complex formation from the membrane inhibits fusion, suggesting that this distance is crucial for atlastin to promote fusion.
Project description:The evolution of tryptophan-to-heme (W/heme) distance distributions extracted from analysis of fluorescence energy transfer kinetics during the refolding of Rhodopseudomonas palustris cytochrome c' reveals dramatic differences between two variants [W32 (Q1A/F32W/W72F) and W72 (Q1A)]. Both W32/heme and W72/heme distance distributions measured at the earliest time point attainable with a continuous-flow mixer (150 mus) confirm that the polypeptide ensemble is not uniformly collapsed and that native structure is not formed. Time-resolved fluorescence spectra indicate that W32 is sequestered from the aqueous solution during the first 700 mus of folding, whereas W72 remains exposed to solvent. The first moment of the W32/heme distance distribution evolves to its native value faster than that of W72, suggesting that the approach of W32 to the heme precedes that of W72.
Project description:T7 DNA primase is composed of a catalytic RNA polymerase domain (RPD) and a zinc-binding domain (ZBD) connected by an unstructured linker. The two domains are required to initiate the synthesis of the diribonucleotide pppAC and its extension into a functional primer pppACCC (de novo synthesis), as well as for the extension of exogenous AC diribonucleotides into an ACCC primer (extension synthesis). To explore the mechanism underlying the RPD and ZBD interactions, we have changed the length of the linker between them. Wild-type T7 DNA primase is 10-fold superior in de novo synthesis compared to T7 DNA primase having a shorter linker. However, the primase having the shorter linker exhibits a two-fold enhancement in its extension synthesis. T7 DNA primase does not catalyze extension synthesis by a ZBD of one subunit acting on a RPD of an adjacent subunit (trans mode), whereas de novo synthesis is feasible in this mode. We propose a mechanism for primer initiation and extension based on these findings.
Project description:RecQ helicases have attracted considerable interest in recent years due to their role in the suppression of genome instability and human diseases. These atypical helicases exert their function by resolving a number of highly specific DNA structures. The crystal structure of a truncated catalytic core of the human RECQ1 helicase (RECQ1(49-616)) shows a prominent ?-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the ?-hairpin is required for the DNA unwinding and Holliday junction (HJ) resolution activity of full-length RECQ1, confirming that it represents an important determinant for the distinct substrate specificity of the five human RecQ helicases. In addition, we found that the ?-hairpin is required for dimer formation in RECQ1(49-616) and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ1(49-616) dimers in solution and demonstrated that dimer formation favours DNA unwinding; even though RECQ1 monomers are still active. Tetramers are instead necessary for more specialized activities such as HJ resolution and strand annealing. Interestingly, two independent protein-protein contacts are required for tetramer formation, one involves the ?-hairpin and the other the N-terminus of RECQ1, suggesting a non-hierarchical mechanism of tetramer assembly.
Project description:The J-domain protein Zuotin is a multi-domain eukaryotic Hsp70 co-chaperone. Though it is primarily ribosome-associated, positioned at the exit of the 60S subunit tunnel where it promotes folding of nascent polypeptide chains, Zuotin also has off-ribosome functions. Domains of Zuotin needed for 60S association and interaction with Hsp70 are conserved in eukaryotes. However, whether the 4-helix bundle (4HB) domain is conserved remains an open question. We undertook evolutionary and structural approaches to clarify this issue. We found that the 4HB segment of human Zuotin also forms a bundle of 4 helices. The positive charge of Helix I, which in Saccharomyces cerevisiae is responsible for interaction with the 40S subunit, is particularly conserved. However, the C-termini of fungal and human 4HBs are not similar. In fungi the C-terminal segment forms a plug that folds back into the bundle; in S. cerevisiae it plays an important role in bundle stability and, off the ribosome, in transcriptional activation. In human, C-terminal helix IV of the 4HB is extended, protruding from the bundle. This extension serves as a linker to the regulatory SANT domains, which are present in animals, plants and protists, but not fungi. Further analysis of Zuotin sequences revealed that the plug likely arose as a result of genomic rearrangement upon SANT domain loss early in the fungal lineage. In the lineage leading to S. cerevisiae, the 4HB was subjected to positive selection with the plug becoming increasingly hydrophobic. Eventually, these hydrophobic plug residues were coopted for a novel regulatory function-activation of a recently emerged transcription factor, Pdr1. Our data suggests that Zuotin evolved off-ribosome functions twice-once involving SANT domains, then later in fungi, after SANT domain loss, by coopting the hydrophobic plug. Zuotin serves as an example of complex intertwining of molecular chaperone function and cell regulation.
Project description:Many examples of enzymes that have lost their catalytic activity and perform other biological functions are known. The opposite situation is rare. A previously unnoticed structural similarity between the lambda integrase family (Int) proteins and the AraC family of transcriptional activators implies that the Int family evolved by duplication of an ancient DNA-binding homeodomain-like module, which acquired enzymatic activity. The two helix-turn-helix (HTH) motifs in Int proteins incorporate catalytic residues and participate in DNA binding. The active site of Int proteins, which include the type IB topoisomerases, is formed at the domain interface and the catalytic tyrosine residue is located in the second helix of the C-terminal HTH motif. Structural analysis of other 'tyrosine' DNA-breaking/rejoining enzymes with similar enzyme mechanisms, namely prokaryotic topoisomerase I, topoisomerase II and archaeal topoisomerase VI, reveals that the catalytic tyrosine is placed in a HTH domain as well. Surprisingly, the location of this tyrosine residue in the structure is not conserved, suggesting independent, parallel evolution leading to the same catalytic function by homologous HTH domains. The 'tyrosine' recombinases give a rare example of enzymes that evolved from ancient DNA-binding modules and present a unique case for homologous enzymatic domains with similar catalytic mechanisms but different locations of catalytic residues, which are placed at non-homologous sites.